The invention generally relates to angiogenic factors and more particularly to the angiopoietin family of growth factors and to methods of decreasing or inhibiting vascular permeability and/or plasma leakage.
Plasma leakage, a key component of inflammation, occurs in a distinct subset of the microvessels. In particular, in most organs plasma leakage occurs specifically in the venules. Unlike arterioles and capillaries, venules become leaky in response to numerous inflammatory mediators including histamine, bradykinin, and serotonin.
One characteristic of inflammation is the plasma leakage that results from intercellular gaps that form in the endothelium of venules. Most experimental models of inflammation indicate that these intercellular gaps occur between the endothelial cells of postcapillary and collecting venules (Baluk, P., et al., Am. J. Pathol. 1998 152:1463-76). It has been shown that certain lectins may be used to reveal features of focal sites of plasma leakage, endothelial gaps, and finger-like processes at endothelial cell borders in inflamed venules (Thurston, G., et al., Am. J. Physiol, 1996, 271: H2547-62). In particular, plant lectins have been used to visualize morphological changes at endothelial cell borders in inflamed venules of, for example, the rat trachea. Lectins, such as conconavalin A and ricin, that bind focally to inflamed venules reveal regions of the subendothelial vessel wall exposed by gaps that correspond to sites of plasma leakage (Thurston, G., et al., Am J Physiol, 1996, 271: H2547-62).
The properties of the microvessels are dynamic. Chronic inflammatory diseases, for example, are associated with microvascular remodeling, including angiogenesis and microvessel enlargement. Microvessels can also remodel by acquiring abnormal phenotypic properties. In a murine model of chronic airway inflammation, we found that airway capillaries acquire properties of venules, including widened vessel diameter, increased immunoreactivity for von Willebrand factor, and increased immunoreactivity for P-selectin. In addition, these remodeled vessels leak in response to inflammatory mediators, whereas vessels in the same position in the airways of normal mice do not.
Certain substances have been shown to decrease or inhibit vascular permeability and/or plasma leakage. For example, mystixins are synthetic peptides that have been reported to inhibit plasma leakage without blocking endothelial gap formation (Baluk, P., et al., J. Pharmacol. Exp. Ther., 1998, 284: 693-9). Also, the beta 2-adrenergic receptor agonist formoterol reduces microvascular leakage by inhibiting endothelial gap formation (Baluk, P. and McDonald, D. M., Am. J. Physiol., 1994, 266:L461-8).
What factors determine whether a vessel will acquire phenotypic features of venules? One apparent clue came from studies of angiopoietin-1 (Ang1), a ligand for the endothelial cell-specific receptor Tie2. In mice that transgenically overexpress Ang1 in the skin under the keratin-14 promoter (K14-Ang1 mice), microvessels in the position of capillaries have widened vessel diameter, immunoreactivity for P-selectin, and immunoreactivity for von Willebrand factor. Thus, these vessels have phenotypic features of venules.
Chronic inflammation is associated with blood vessel formation and enlargement and changes in vessel phenotype. In mice with chronic airway inflammation, strain dependent differences to the same stimulus have been shown to result in either blood vessel proliferation or enlargement, depending on the host response (Thurston, G., et al., Am. J. Pathol., 1998, 153: 1099-112). Analyses of mouse embryos deficient in the TIE-2 receptor illustrate its importance in angiogenesis, particularly for vascular network formation in endothelial cells. Sato, T. N., et al., Nature 376:70-74 (1995). In the mature vascular system, the TIEs could function in endothelial cell survival, maintenance and response to pathogenic influences.
It has been suggested that the TIE receptors play roles in endothelial cell determination, proliferation, differentiation and cell migration and patterning into vascular elements. The predominant expression of the TIE receptors in vascular endothelia suggests that TIE plays a role in the development and maintenance of the vascular system. The TIE receptors are also expressed in primitive hematopoietic stem cells, B cells and a subset of megakaryocytic cells, thus suggesting the role of ligands which bind these receptors in early hematopoiesis, in the differentiation and/or proliferation of B cells, and in the megakaryocytic differentiation pathway. Iwama, et al. Biochem. Biophys. Research Communications 195:301-309 (1993); Hashiyama, et al. Blood 87:93-101 (1996), Batard, et al. Blood 87:2212-2220 (1996).
An angiogenic factor, which was originally called TIE-2 ligand-1 (TL1) but is also referred to as angiopoietin-1 (Ang1), has been identified that signals through the TIE-2 receptor and is essential for normal vascular development in the mouse. By homology screening, an Ang1 relative has been identified and called TIE-2 ligand-2 (TL2) or angiopoietin-2 (Ang2). Ang2 is a naturally occurring antagonist for Ang1 and the TIE2 receptor. For a description of the cloning and sequencing of TL1 (Ang1) and TL2 (Ang2) as well as for methods of making and uses thereof, reference is hereby made to PCT International Publication No. WO 96/11269 published Apr. 18, 1996 and PCT International Publication No. WO 96/31598 published Oct. 10, 1996 both in the name of Regeneron Pharmaceuticals, Inc.; and S. Davis, et al., Cell 87: 1161-1169 (1996) each of which is hereby incorporated by reference. Ang1* is a mutant form of angiopoietin-1 that comprises the N-terminal domain of angiopoietin-2 fused to the coiled-coil domain and the fibrinogen domain of angiopoietin-1 and that has a Cys to Ser mutation at amino acid 245 (See PCT International Publication No. WO 98/05779 published Feb. 12, 1998 in the name of Regeneron Pharmaceuticals, Inc. which is hereby incorporated by reference). Ang1* has been shown to be a potent agonist for the Tie-2 receptor.
Including the above-described angiopoietins, applicants have identified a family of several related angiogenic factors. These have been designated TIE-2 ligand-1 (TL1) also referred to as angiopoietin-1 (Ang1); TIE-2 ligand-2 (TL2) or angiopoietin-2 (Ang2); Tie ligand-3 (TL3) and Tie ligand-4 (TL4). For descriptions of the structure and functional properties of these four related factors, reference is hereby made to the following publications, each of which is hereby incorporated by reference: U.S. Pat. No. 5,643,755, issued Jul. 1, 1997 to Davis, et al.; U.S. Pat. No. 5,521,073, issued May 28, 1996 to Davis, et al.; U.S. Pat. No. 5,650,490, issued Jul. 22, 1997 to Davis, et al.; U.S. Ser. No. 08/348,492, filed Dec. 2, 1994, now allowed, date of allowance Aug. 29, 1997; U.S. Ser. No. 08/418,595, filed Apr. 6, 1995, now allowed, date of allowance Nov. 26, 1996; U.S. Ser. No. 08/665,926, filed Jun. 19, 1996, now allowed, date of allowance Dec. 9, 1997; PCT International Application No. PCT/US95/12935, filed Oct. 6, 1995, published on Apr. 18, 1996, with Publication No. WO 96/11269; and PCT International Application No. PCT/US96/04806, filed Apr. 5, 1996, published on Oct. 10, 1996, with Publication No. WO96/31598, both PCT applications in the name of Regeneron Pharmaceuticals, Inc.
The angiopoietins can be structurally divided into three domains: an N terminal region lacking in homology to any known structures; an alpha-helical rich coil-coil segment similar to motifs found in many proteins that seem to promote multimerization; and a xe2x80x9cfibrinogen-like domainxe2x80x9d thus dubbed because it is distantly related to a domain first found in fibrinogen but now noted to be in many other proteins (Davis, S. et al., (1996) Cell 87: 1161-1169). The fibrinogen-like domain represents the most conserved region of the angiopoietins, and recent studies indicate that it comprises the receptor-binding portion of an angiopoietin. In addition, all the information that determines whether an angiopoietin is an agonist or an antagonist appears to reside within the fibrinogen-like domain. For example, when chimeric molecules are made in which the fibrinogen-like domains of angiopoietin-1 and angiopoietin-2 are swapped, agonistic or antagonistic abilities track with the fibrinogen-like domains. The N-terminal and coil-coil regions appear to serve mainly to multimerize the fibrinogen-like domains, which apparently must be clustered to be active. In fact, the N-terminal and coil-coil regions can be substituted for by alternative motifs that allow clustering. Thus, the activities of angiopoietin-1and angiopoietin-2 can be precisely mimicked by surrogates in which the fibrinogen-like domains (FD) of these factors are fused to the constant region of an antibody, resulting in FD-Fc fusions, which can then be clustered using secondary antibodies directed against the Fc. For example, TIE-2 ligand 1 contains a xe2x80x9ccoiled coilxe2x80x9d domain and a fibrinogen-like domain. The fibrinogen-like domain of TIE-2 ligand 2 is believed to begin on or around the same amino acid sequence as in ligand 1 (FRDCA). The fibrinogen-like domain of TIE ligand-3 is believed to begin on or around the amino acid sequence which is encoded by nucleotides beginning around position 929 as set forth in FIGS. 6A-6B in International Publication No. WO 97/48804 published Dec. 24, 1997. Multimerization of the coiled coil domains during production of the ligand hampers purification. As described in Example 7 of International Publication No. WO 97/48804, Applicants have discovered, however, that the fibrinogen-like domain comprises the TIE-2 receptor binding domain. The monomeric forms of the fibrinogen-like domain do not, however, appear to bind the receptor. Studies utilizing myc-tagged fibrinogen like domain, which has been xe2x80x9cclusteredxe2x80x9d using anti-myc antibodies, do bind the TIE-2 receptor. [Methods of production of xe2x80x9cclustered ligands and ligandbodies are described in Davis, et al. Science 266:816-819 (1994)]. Based on these finding, Applicants produced xe2x80x9cligandbodiesxe2x80x9d which comprise the fibrinogen-like domain of the TLE-2 ligands coupled to the Fc domain of IgG (xe2x80x9cfFc""sxe2x80x9d). These ligandbodies, which form dimers, efficiently bind the TIE-2 receptor. Accordingly, the present invention contemplates the production of TIE ligand-3 or TIE ligand-4 ligandbodies which may be used as targeting agents, in diagnostics or in therapeutic applications, such as targeting agents for tumors and/or associated vasculature wherein a TIE antagonist is indicated.
In order to test whether the fibrinogen-like domain (F-domain) of the TIE-2 ligands contained TIE-2 activating activity, expression plasmids were constructed which deleted the coiled-coil domain, leaving only that portion of the DNA sequence encoding the F-domain (beginning at about nucleotide 1159, amino acid residue ARG284) This mutant construct was transiently transfected into COS cells. The supernatant containing the recombinant protein was harvested. The TL1/F-domain mutant was tested for its ability to bind the TIE-2 receptor. The results showed that, as a monomer, the TL1/F domain mutant was not able to bind TIE-2 at a detectable level. However, when the TL1/F-domain monomer was myc-tagged and subsequently clustered with an antibody directed against the myc tag, it did exhibit detectable binding to TIE-2 However, the antibody clustered TL1/F-domain mutant was not able to induce phosphorylation in a TIE-2 expressing cell line. FIG. 3 of International Publication No. WO 97/48804 shows a schematic representation of the F-domain construct and its binding ability plus and minus antibody clustering. For a general description of the production and use of FD-Fc fusions, see International Publication Number WO 97/48804 published Dec. 24, 1997. Using these techniques, one of skill in the art would be able to similarly make FD Fc fusions using the fibrinogen-like domain of an Angiopoietin family member. One practical advantage of such surrogates is that native angiopoietins can be difficult to produce recombinantly, while the surrogates can be more easily produced.
The absence of Ang1 causes severe vascular abnormalities in the developing mouse embryo (C. Suri, et al., Cell, 1996, 87: 1171-1180). Ang1 and Ang2 have been described as naturally occurring positive and negative regulators of angiogenesis. Positive or negative regulation of TIE2 is likely to result in different outcomes depending on the combination of simultaneously acting angiogenic signals.
The angiopoietins and members of the vascular endothelial growth factor (VEGF) family are the only growth factors thought to be largely specific for vascular endothelial cells. Targeted gene inactivation studies in mice have shown that VEGF is necessary for the early stages of vascular development and that Ang-1 is required for later stages of vascular remodeling. It has been reported that transgenic overexpression of Ang-1 in the skin of mice produces larger, more numerous, and more highly branched vessels, however the characteristics of the resultant vessels are largely unknown (Suri, C., et al., Science, 1998, 282:468-171). The present invention is the result of applicants""efforts to examine these vessels in greater detail by, among other studies, assaying for vessel permeability/plasma leakage.
The present invention provides for a method of decreasing or inhibiting vascular permeability and/or plasma leakage in a mammal comprising administering to the mammal a TIE-2 receptor activator. By way of example, but not by way of limitation, the vascular permeability/plasma leakage may be produced by an inflammatory agent or by injury. In one embodiment of the invention, the mammal is a human. By vascular permeability, what is meant is any process that leads to leakage or extravasation of plasma. including, but not limited to, increased endothelial permeability. See for example, McDonald, D. M., et al., Microcirculation 6: 7-22 (1999); Feng, D., et al., Microcirculation 6: 23-44 (1999); and Michel, C. C., and Neal, C. R., Microcirculation 6: 45-54 (1999) each of which is incorporated by reference herein in its entirety.
The invention further provides for a method wherein the TIE-2 receptor activator is Angiopoietin-1, or a fragment or derivative thereof capable of activating the TIE-2 receptor.
The invention also provides for a method wherein the TIE-2 receptor activator is an activating antibody, or a fragment or derivative thereof capable of activating the TIE-2 receptor.
The invention further provides for a method wherein the TIE-2 receptor activator is a small molecule, or a fragment or derivative thereof capable of activating the TIE-2 receptor.
The invention also provides for a method of decreasing or inhibiting vascular permeability and/or plasma leakage in a mammal comprising administering to the mammal an Ang-2 inactivator such as an anti-Ang-2 neutralizing antibody. In one embodiment of this invention, the mammal is a human. The invention also provides for a method wherein the anti-Ang-2 neutralizing antibody is a monoclonal antibody. The invention further provides for a method wherein the anti-Ang-2 neutralizing antibody is a polyclonal antiserum. Alternatively the Ang-2 inactivator could be a small molecule.
The invention also provides for a composition comprising an Ang-2 inactivator in a vehicle, as well as a method of regulating angiogenesis in a patient comprising administering to the patient an effective amount of the composition. In one embodiment, the Ang-2 inactivator is a small molecule. The invention also provides for a composition comprising an anti-Ang-2 neutralizing antibody in a vehicle, as well as a method of regulating angiogenesis in a patient comprising administering to the patient an effective amount of the composition.